1. Field of the Invention
The present invention relates generally to methods and compositions useful for immobilizing oligonucleotides and other biological polymers on surfaces. The immobilized biological polymers, which can be, for example, oligonucleotides or polypeptides, are useful in a variety of screening and assay methodologies. The present invention has applications in the fields of molecular biology, biochemistry, pharmacology, and medical diagnostic technology.
2. Description of Related Art
Many different methods are known for attaching biological molecules to solid supports. See generally, Affinity Techniques, Enzyme Purification: Part B, Meth. Enz. 34 (ed. W. B. Jakoby and M. Wilchek, Acad. Press, N.Y. 1974) and Immobilized Biochemicals and Affinity Chromatography, Adv. Exp. Med. Biol. 42 (ed. R. Dunlap, Plenum Press, N.Y. 1974), incorporated herein by reference. The patent literature also describes a number of different methods for attaching biological molecules to solid supports. For example, U.S. Pat. No. 4,282,287 describes a method for modifying a polymer surface through the successive application of multiple layers of biotin, avidin, and extenders. U.S. Pat. No. 4,562,157 describes a technique for attaching biochemical ligands to surfaces by attachment to a photochemically reactive arylazide. Irradiation of the azide creates a reactive nitrene that reacts irreversibly with macromolecules in solution resulting in the formation of a covalent bond. The high reactivity of the nitrene intermediate, however, results in both low coupling efficiencies and many potentially unwanted products due to nonspecific reactions.
U.S. Pat. No. 4,681,870 describes a method for introducing free amino or carboxyl groups onto a silica matrix; the groups may subsequently be covalently linked to a protein in the presence of a carbodiimide. Also, U.S. Pat. No. 4,762,881 describes a method for attaching a polypeptide chain to a solid substrate by incorporating a light-sensitive unnatural amino acid group into the polypeptide chain and exposing the product to low-energy ultraviolet light.
A variety of techniques have also been developed for attaching oligonucleotides to surfaces. For example, U.S. Pat. No. 4,542,102 describes a method employing a photochemically active reagent (e.g., a psoralen compound) and a coupling agent, which attaches the photoreagent to the substrate. Photoactivation of the photoreagent binds a nucleic acid sequence to the substrate to give a surface-bound probe for a complementary oligonucleotide. However, this method has low quantum yields in protic solvents, lacks spatial directability, and relies upon initial affinity between the photoreagent and nucleic acids to bind the nucleic acids to the surface prior to photoactivation.
U.S. Pat. No. 4,937,188 describes methods for forming maleimide-thiol linkages between a solid support and a molecular tether. In one example, thiol groups on sepharose are reacted with maleimide groups on an RNA polymer, which serves as the tether, and the RNA polymer is reacted with a protein, also via a maleimide-thiol linkage. In another example, maleimide groups immobilized on a solid support are reacted with sulfhydryl RNAse forming RNAse-agarose. U.S. Pat. No. 5,011,770 describes the use of a maleimide-thiol linkage to bind an enzyme label to a binding protein, which in turn binds to single stranded DNA. The patent describes that the binding protein can also be attached to a solid support. The maleimide-thiol linkage is thus spatially separated from the actual linkage between the peptide and the solid support.
PCT patent publication No. 90/07582 describes polyacrylamide supports on which are immobilized oligonucleotides. The oligonucleotides are derivatized at the 5'-terminus with thiol groups that are reacted with bromoacetyl groups on the polyacrylamide support during the immobilization process. PCT patent publication No. 91/00868 discloses solid supports on which oligonucleotides are immobilized via a 5'-dithio linkage.
The immobilization of biological polymers on solid supports has also had significant impact on drug discovery and medical diagnostic methods. One important invention in these fields is described in U.S. Pat. No. 5,143,854 an in Ser. Nos. 624,120, filed Dec. 6, 1990, and 805,727, filed Dec. 6, 1991, and in PCT patent publication No. 90/15070 to Pirrung et al., each of which is incorporated herein by reference. In brief, the invention provides methods and reagents for synthesizing very large numbers of different compounds, particularly biological polymers, in distinct and spatially-addressable locations in a very small area on a substrate. Another description of the invention is found in Fodor et al., 15 Feb. 1991, Science 251:767-773, and the integration of the invention with other drug discovery methods is described in Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271-280. A related method uses a photoactivateable derivative of biotin as the agent for immobilizing a biological polymer of interest onto a solid support; see U.S. Pat. No. 5,252,743, and PCT patent publication No. 91/07087 to Barrett et al., each of which is incorporated herein by reference.
Recent approaches to genetic analysis are increasingly placing importance on performing parallel hybridizations in an array format. Applications of the parallel hybridization format include generating diagnostic arrays for tissue typing or diagnosis of genetic disorders (see PCT patent publication No. 89/11548, incorporated herein by reference), DNA sequencing by hybridization, DNA fingerprinting, and genetic mapping (see U.S. patent application Ser. Nos. 624,114, now abandoned and 626,730, filed Dec. 6, 1990, each of which is incorporated herein by reference; see also Khrapko et al., 1991, J. DNA, Seq. Map. 375-388). In these applications of probe arrays, the information content of the array increases as the number of probes is increased. The size limit of the array is dictated by the ability to automate and miniaturize the fabrication of the array.
Thus, there exists a need for improved methods for attaching a broad range of anti-ligands to predefined regions of a solid support surface. The methods should efficiently provide stable attachment of selected polymers to the activated surface regions, yet attachment should be restricted to the activated regions. The present invention fulfills these and other needs.